HuSdL TM Phage Display Human Single Domain Antibody Library · After 30 min incubation on ice,...

HuSdLTM

Phage Display

Human Single Domain

Antibody Library Kit

User Manual

Cat. No. HuSdLTM

Published on Feb 19, 2011

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Phage Display Human Single Domain Ant ibody Library User Manual

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I. List of Components

HuSdLTM: Phage Display Human Single Domain Antibody Library. ~1.0×1012 recombinant phages supplied

in 100 μL PBS with 15% glycerol. Complexity=1.5×109 transformants; ampicillin resistant.

E. coli TG1 host strain: K12 D(lac-pro), supE, thi, hsdD5/F’*traD36, proAB, lacIq, lacZDM15+.

E. coli HB2151 host strain: K12 D(lac-pro), ara, nalr, thi/F’*proAB, lacIq, lacZDM15].

M13K07 helper phage: ~1.0×1011pfu phages (~1.0×1012 pfu/mL) supplied in 100μL TBS with 15% glycerol.

Kanamycin resistant.

The sequencing primers for HuSdLTM Human Single domain Antibody Library:

L1: 5'-TGGAATTGTGAGCGGATAACAATT-3'

S6: 5'-GTAAATGAATTTTCTGTATGAGG-3'

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II. Introduction & Library Overview

Principle of phage display

In a phage display library, a variety of peptides, small antibodies [e.g. scFv and Fab] or proteins are

displayed on the surface of filamentous phage [M13, fd, and f1 strains] or lambda phage as fusion

proteins with one of the coat proteins of the phage virions, while the genetic materials encoding the

peptides/proteins are housed within the virion. Using a binding-based process called bio-panning [Figure

1], a small number of phages that display proteins specifically binding to a target of interest can be

rescued from a phage library that usually displays a repertoire of many billions of unique

peptides/proteins. Finally, the peptides/proteins displayed by the selected phages can be identified by

phage amplification and DNA sequencing.

Figure 1. Illustration of phage display cycle

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Figure 2. Phage display of antibody fragments.

The antibody fragment (such as variable heavy chain, VH, in red) is displayed as a fusion with the

terminal phage geneIII protein (green). Both proteins are encoded by the phagmid DNA (magnified) and

expressed from a common promoter. The major gene VIII coat protein is shown in white (other phage

proteins not shown).

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HuSdLTM Phage Display Human Single Domain Antibody Library

HuSdLTM Human Single Domain Antibody Library was constructed based on camelised human VH3. At

the same time, the human HCDR3 was elongated and sequence-randomized. The genes and

pCDisplay-5TM vector were digested by restriction enzyme SfiI and Not I then ligated together. The

recombinant constructs were electro-transformed into competent E. coli TG1 cells. Finally, the Human

Single Domain Antibody Library with a calculated diversity of 1.5×109 was constructed.

Our Human Single Domain Antibody Library produces camelised human antibodies that have a human

origin, thus the lowest immunogenic potential in humans, especially for long-term and multiple-dose

administration. This library was preselected based on the thermostability and expressibility [in E. coli.]

of the displayed antibodies. Specifically, in the library, the antibody repertoire was heat-treated to

remove clones that could not withstand heat-induced aggregation. The genetic codons used to encode

the antibody binders are also optimized of bacterial preference.

Using the protocol provided in this manual, specific single domain antibody binders can be selected very

rapidly (generally within two months). This library is an invaluable antibody resource for isolating human

single domain antibody binders for research, diagnostic and therapeutic applications.

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III. Materials and Methods for Using the Library

Materials

HuSdLTM: Phage Display Human Single Domain Antibody Library, product of Creative Biolabs.

E. coli TG1 host strain: From Stratagene TG1 is a suppressor strain, in which the amber stop codon

would not be recognized and the single domain antibodies would be fused with phage coat protein III

and displayed on the surface of phage virions.

E. coli HB2151 host strain: From GE heathcare. HB2151 is a non-suppressor strain, in which the amber

stop codon would be recognized and the soluble single domain antibodies would be produced.

M13K07 helper phage: GE Healthcare.

Media and solution

LB Medium: Per liter: 10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl. Autoclave 20 minutes at 121°C

LB Plates: LB medium + 15 g/L agarose

Top agar: as for LB medium, but use 7 g/L Bacto-agar. Before use, melt and cool to 50°C

2TY medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, made up to 1 L in deionized H2O, and

adjusted to pH 7.0 with 1 M NaOH. Autoclave

2TY-G: 2TY containing 1%(w/v) glucose; 2TY-A: 2TY containing 100ug/mL ampicillin (AMP)

2TY-AG: 2TY containing 100ug/mL ampicillin (AMP) and 1%(w/v) glucose

2TY-AK: 2TY containing 100 ug/mL ampicillin, 50 ug/mL kanamycin

TYE agar plates: Add 15 g agar to 1 L 2TY medium, autoclave, when cool, add glucose to 1% (w/v) and

AMP

PBS: Per liter: 8 g NaCl, 0.2 g KCl, 1.7 g Na2HPO4, 0.163 g KH2PO4, pH to 7.4 with HCl

Blocking buffer: 0.1M NaHCO3 (pH 8.6), 5 mg/mL BSA, 0.02% NaN3. Filter sterilize, store at 4°C

Coating Buffer: 0.1M NaHCO3 (pH 8.6).

Conjugated second antibody:

HRP-conjugated anti-M13 monoclonal antibody from mouse: GE Healthcare

HRP conjugate of anti-c-Myc Tag: Pierce

EIA/RIA strip well 96-well plates: Corning, USA

Substrate: OPD and H2O2 from sigma

5*PEG/NaCl: Dissolve in water: 200 g of polyethylene glycol-8000, 150 g of NaCl(2.5M). Bring volume to

1 liter; stir until dissolved. Filter through a 0.8 µm filter

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Library Screening

1. Maintenance of Bacterial Strains and Viruses

E. coli TG1 host strain: may be stored at 4 °C for up to two weeks on minimal medium; Prepare

glycerol stocks (add sterile 80% glycerol to stationary phase cells, mix well) store at -70°C for long

term storage.

E. coli HB2151 host strain: may be stored at 4 °C for up to two weeks on minimal medium; Prepare

glycerol stocks (add sterile 80% glycerol to stationary phase cells, mix well) store at -70°C for long-

term storage.

M13K07 helper phage: This helper phage carries the kanamycin resistance gene for antibiotic

selection. Stocks of the phage can be stored at –80°C in 7% (v/v) DMSO indefinitely, or for up to 6

months at 4°C.

HuSdLTM: can be stored indefinitely at 4°C (in 1% BSA in PBS–0.01% [w/v] NaN3 ). Libraries should be

freshly amplified before panning. Keep plasmid DNA stocks of the primary library and/or glycerol

stocks of bacteria containing the phagemid library.

2. Amplification of M13KO7 helper phage

1) Prepare helper phage by infecting log-phase TG1 bacterial cells with M13K07 phage at different

dilutions for 30 min at 37°C and plating in top agar onto 2TY plates.

2) Inoculate a small plaque in 3 mL liquid 2TY medium. Add 30 μL overnight culture of TG1 and grow

for 2 h at 37°C.

3) Dilute the culture in 1 L 2TY medium and grow for 1 h. Add kanamycin to 50μg/mL and grow for 16

h at 37°C.

4) Remove cells by centrifugation (10 min at 5,000g) and precipitate phage from the supernatant by

addition of 0.25 vol of phage precipitant. After 30 min incubation on ice, collect the phage particles

by centrifugation during 10 min at 5,000g. Re-suspend the pellet in 5 mL PBS and sterilize through a

0.22-um filter.

5) Titrate the helper phage by determining the number of plaque-forming units (pfu) on 2TY plates

with top-agar layers containing 100 μL TG1 (saturated culture) and various dilutions of phage.

Dilute the phage stock solution to 1 × 1013 pfu/mL and store in small aliquots at –20°C.

3. Panning

1) Coat the target antigen (1-10μg/ml) in the panning plates in the coating buffer. Incubate at 37°C for

two hours.

2) Block panning plates (NUNC) with the blocking buffer at 4 °C overnight.

3) Wash blocked wells 6 times with 0.1%PBST (PBS with 0.1% Tween 20(V/V))

4) Mix equal volumes of the phage library and 4% PBSM (PBS containing 4% milk) in a total volume of

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0.5 mL.

5) During the first round of screening, the number of phage particles should be around 100× greater

than the library size (e.g., 1012 pfu for a library of 1010 clones). Diversity drops to 106 after the first

round and thus there is no such a requirement in the subsequent rounds of screening. Incubate 30

min at room temperature to block the binding sites.

6) Add input phage mix into panning wells and incubate at room temperature for 60 min.

7) Wash 10-20 times with PBSMT (PBS containing 2% milk and a certain percent of Tween-20).

8) Elute the phage by adding 200 μL either acidic eluting buffer or competitive eluting buffer and

incubate 5 min at room temperature. Transfer the supernatant containing the phages to a new

tube.

9) Infect a fresh exponentially growing culture of Escherichia coli TG1 with the eluted phages and

amplify half of them for further rounds of selection.

a) Inoculate 500 mL 2TY-G with the library glycerol stock and incubate at 37°C with shaking at

250 rpm until the optical density at 600 nm reaches 0.8–0.9.

b) Add M13KO7 helper phage to a final concentration of 5 ×109 pfu/mL, and incubate for 30 min

at 37°C without shaking, then for 30 min with gentle shaking (200 rpm), to allow phage

infection.

c) Recover the cells by centrifugation at 2,200g for 15 min and re-suspend the pellet in the same

volume of 2TY-AK. Incubate overnight at 30°C with rapid shaking (300 rpm).

d) Pellet the cells by centrifugation at 7,000g for 15 min at 4°C and recover the supernatant

containing the phage into pre-chilled 1-L bottles.

e) Add 0.3 vol of phage precipitant. Mix gently and allow the phage to precipitate for 1 h on ice.

f) Pellet the phage by centrifuging twice at 7,000g for 15 min in the same bottle at 4°C. Remove

as much of the supernatant as possible and re-suspend the pellet in 8 mL PBS.

g) Re-centrifuge the phage in smaller tubes at 12,000g for 10 min and recover the phage via the

supernatant. Ensure that any bacterial pellet that appears is left undisturbed.

h) Finally, titer phage stocks by infecting TG1 cells with dilutions of phage stock, plating to

2TY-AG, incubation, and enumeration of the numbers of ampicillin resistant colonies that

appear. The phages can then be stored in aliquots at 4°C for long periods, ready for screening.

4. Phage ELISA

1) Prepare single clones of antibody-displaying phages

a) Inoculate single clones of the eulate from the 4th round into 5 mL of 2YT-AG medium and incubate

at 37°C overnight.

b) Prepare the glycerol stock for each clone with the overnight culture. Inoculate 100μL of overnight

culture into 20mL of 2YT-AG medium. Grow for a few hours at 37°C until the optical density at

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OD600 reach 0.4-0.5.

c) Add M13KO7 helper phage at a multiplicity of infection of 20 (i.e., the number of phage

particles/host cell). Infect the cells by incubating 30 min at 37°C without shaking and another 30

min with shaking.

d) Collect infected cells by centrifugation (10 min at 5,000g). Re-suspend in 2YT-AK and grow the

culture for 16 h at 30°C.

e) Precipitate phage particles from the supernatant as described above. Re-suspend the phage pellet

in 1mL PBS and remove cellular debris by centrifugation (10 min at 5,000g).

f) To remove Ab fragments not associated to phage particles, carry out a second precipitation.

Re-suspend the phage pellet in 250 μL PBS, clarify again by centrifugation.

2) Phage ELISA by coating the target directly

a) Coat 100 μL of non-biotinylated target (10μg/mL) in coating buffer by incubating at 4°C overnight

b) Shake out the coating solution and wash once with the washing buffer. Block all wells with 250 μL

of blocking buffer. In order to test the binding of each selected sequence to BSA-coated plastic

surface, enough uncoated wells should also be blocked. Incubate the blocked plates 1-2 hours at

4°C.

c) Shake out the blocking buffer and wash the plate 6 times with the washing buffer.

d) Add 100 μL of phage solution in washing buffer per well. Incubate at room temperature for 1–2

hours.

e) Wash 6 times with the washing buffer. Dilute HRP-conjugated anti-M13 antibody (GE healthcare)

1:5,000 in blocking buffer. Add 100 μL of diluted conjugate per well and incubate at room

temperature for 1 hour.

f) Wash 6 times with the washing buffer. Prepare the HRP substrate solution as follows: a stock

solution of OPD can be prepared in advance by dissolving 22 mg OPD (Sigma) in 100 mL of 50 mM

sodium citrate, pH 4.0. Filter sterilize and store at 4°C. Immediately prior to the detection step, add

36 μL 30% H2O2 to 21 mL of OPD stock solution.

g) Add 100 μL of substrate solution per well and incubate at room temperature for 30 minutes.

h) Read plates using a microplate reader set at 490nm.

5. Soluble domain antibody ELISA

Since HB2151 is a non-suppressor strain, in which the amber stop codon between domain antibody gene

and gene III can be recognized and the soluble domain antibody can be secreted into the periplasm.

1) Soluble expression in periplasm of HB2151 cells

a) Pick a single colony of HB2151 from a LB plate and grow overnight at 37°C in 2TY.

b) Add 100 μL HB2151 culture to 50 mL 2TY and grow at 37°C (with shaking) to an OD600 of 0.6–0.8.

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c) Infect HB2151 with phages of positive clones identified by phage ELISA. Inoculate 1 μL of phages

into 200-μL aliquots of the log-phase HB2151 culture. Incubate for 30 min at 37°C (no shaking).

Plate onto TYE plates and incubate overnight at 37°C.

d) Inoculate a single colony into 5mL aliquot of 2TY-AG growth medium and grow overnight at 37°C

(with gentle shaking).

e) Take a 250 μL aliquot of overnight culture from each well and transfer to 25 mL 2TY growth

medium. Grow the cultures at 37°C until OD600 is approx 0.6.

f) Add IPTG to 1mM and continue to grow 20-24h at 30°C.

g) Collect cells via centrifugation at 3,500g for 10 min. Release soluble domain antibody by

ultra-sonication of cell suspensions in 1/10 volume of PBS

h) Centrifuge at 9,000g for 10 min and collect the supernatant containing soluble domain antibody.

2) Soluble domain antibody ELISA

a) Coat the wells of an ELISA plate with the desired Ag (100 μL, 1-10μg/ mL) in coating buffer.

b) Wash the wells 3× with 200 μL PBS.

c) Block the wells with 200 μL PBSM for 2 h at room temperature.

d) Discard the block solution and add 100 μL of supernatant containing soluble domain antibody.

Incubate for 2 h at room temperature.

e) Wash the wells 3× with 200 μL PBST.

f) Add 100 μL of HRP conjugate of anti-c-Myc Tag Ab diluted in PBSM to each well and incubate for 1

h at room temperature

g) Wash the wells 3× with 200 μL PBST.

h) Develop the HRP reaction using OPD and read optical density at 490nM

6. DNA Sequencing

1) Inoculate 2μL glycerol stock TG1 bacterial cells harboring the plasmid for each positive clone

(determined by phage ELISA and/or soluble ELISA) into 5mL LB-A medium (LB medium added with

100μg/mL ampicillin). Grow overnight at 37°C with shaking.

2) Isolate plasmids for each positive clone from bacteria using the commercialized Plasmid Isolation

Kit (Such as Qiagen Miniprep kit).

3) Conduct DNA sequencing using “L1” and “S6” as the primers.

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IV. Appendix:

1. Schematic map of pCDisplay-5TM (HuSdL

TM was constructed by inserting VH into pCDisplay-5

TM)

Pel B leader sequence Sfi I Not I

Atgaaaaaattattattcgcaattcctttagtggtacctttctatgcggcccagccggccatggcc---------------- VH-------------gcggccgca

Myc SV5

gaacaaaaactcatctcagaagaggatctgaattcggccgcacattatacagacatagagatgaaccgacttggaaag

2. One typical sequence of a single domain antibody:

DNA sequence

5’-Caggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgcgtctctcctgtgcagcctccggat

ttacgcttagcgatgacgatatgggctgggtccgccaggctccagggcagggtctagagtgggtatcagccattcggatgag

taacggtagcacatactacgcagactccgtgaagggccggttcaccatcttccgtgacaattccaagaacacgctgtatctg

caaatgaacagcctgcgtgccgaggacaccgcggtatattattgcgcgagtgttgagccgtttgacgacaagctggagtctt

ggggtcagggaaccctggtcaccgtctcgagc-3’

Protein Sequence

CDR1 CDR2

QVQLLESGGGLVQPGGSLRLSCAASGFTLSDDDMGWVRQAPGQGLEWVSAIRMSNGSTYYADSVKGRFTIFRDNSKNTLYLQ

CDR3

MNSLRAEDTAVYYCASVEPFDDKLESWGQGTLVTVSS

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References

1. C. F. Barbas III, D. R. Burton, J. K. Scott, and G. J. Silverman, (2001) Phage Display: A laboratory

manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

2. O'Brien P.M. Aitken R. Totowa, (2001) Antibody phage display: methods and protocols. In: Methods

in Molecular Biology, Vol 178. Humana Press, NJ.

3. Sachdev S. Sidhu, (2005) Phage Display in Biotechnology and Drug Discovery. CRC Press/Taylor &

Francis Group, Boca Raton.

Contact Information

For more information or technical assistance, please call, write, fax, or email to the following address:

Creative BioLabs

45-16 Ramsey Road

Shirley, NY 11967

USA

Tel: (631)-871-5806, Fax: (631)-207-8356

E-mail: [email protected]

Web: www.creative-biolabs.com

[© 2011 Creative BioLabs, CD Inc. All rights reserved]

mailto:[email protected]

http://www.creative-biolabs.com/

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